Integrated method for collection and maintenance of detectability of a plurality of microbiological agents in a single clinical sample and for handling a plurality of samples for reporting a sum of diagnostic results for each sample

ABSTRACT

A method and kit related thereto are described for the collection and maintenance of detectability of a plurality of species of microbiological agents in a single clinical sample as well as an integral method for handling a plurality of the samples and managing information associated therewith for reporting a sum of diagnostic results for each sample.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/343,822, filed Jan. 31, 2006, which claims the benefit under 35U.S.C. 119(e), to U.S. Provisional Application No. 60/651,688, filed onFeb. 10, 2005, the entire contents of which are hereby incorporated byreference, to U.S. Provisional Application No. 60/654,485, filed on Feb.18, 2005, the entire contents of which are hereby incorporated byreference, and to U.S. Provisional Application No. 60/654,729, filed onFeb. 18, 2005, the entire contents of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the field of clinical diagnostic services,i.e., in the industry of identification, characterization,quantification of biological agents associated with disease conditions.The present invention is particularly directed toward a method andmaterials for the collection and maintenance of detectability of aplurality of species of microbiological agents selected from the groupconsisting of bacteria, fungi, viruses, and protozoa in a singleclinical sample and managing information associated with a plurality ofsamples for reporting a sum of diagnostic results for each sample.

2. Description of the Related Art

Clinical diagnostics provide an essential aid to the physician for thediagnosis and monitoring of numerous pathologies and infectiousdiseases. Rapid and accurate identification of causative agents of amyriad of different human pathophysiological conditions is a paramountrequisite to effective treatment.

A biological sample generally is taken from the patient, most often atthe request of a physician, and sent to a medical laboratory foranalysis to establish or confirm a diagnosis of clinical symptoms. Aphysician may suspect a particular causative agent upon physicalexamination. However, certain symptoms may be characteristic of aplethora of different causative agents. Therefore, due to misdiagnosesof causative agents, patients may be treated non-efficaciously.Moreover, in other instances, a physician may request a certaindiagnostic test to be performed on a clinical specimen wherein the testsubsequently produces a negative result. Then, of course, furtherclinical samples and diagnostic testing are required. In many instances,due to the lack of timely and accurate diagnoses, patients' originalconditions progress to the further detriment of treatability and to thewell-being of the patient. Accurate clinical diagnosis is critical tospecifically identify causative agents in a timely manner which mediatespathophysiological conditions. Accordingly, a need indeed exists formaterials and a method, for example, to collect an accurate clinicalsample, e.g. a “snapshot,” representative of vaginal flora, i.e., acertain gynecological microbiological environment, and maintain thedetectability of a plurality of species in a single gynecologicalsample.

Diagnostic kits are available, for example, capable of detecting severalspecific species. However, in many instances current diagnostic productsand services are inadequate to identify the causative agent or areinoperable under clinical circumstances.

Advances in the detection of C. trachomatis and N. gonorrhoeae, forexample, have included the development of nucleic acid amplificationtests from cervical as well as urine samples. The Roche COBAS AMPLICOR™CT/NG Test, for example, is an in vitro multiplex diagnostic test thatcan detect either or both Chlamydia trachomatis or Neisseria gonorrhoeaein endocervical or urethral swabs and/or urine samples. The COBASAMPLICOR™ Analyzer is an instrument which automates amplification anddetection of the PCR process. The test utilizes polymerase chainreaction (PCR) nucleic acid amplification and nucleic acid hybridization(Roche Diagnostic Systems, Branchburg, N.J.). APTIMA COMBO 2 Assay is aGen-Probe nucleic acid amplification test that uses target capture forin vitro qualitative detection and differentiation of rRNA from C.trachomatis and N. gonorrhoeae in endocervical and male urethral swabspecimens and in urine specimens. The assay uses target capture (TC),Transcription-Mediated Amplification (TMA) and Dual Kinetic Assay (DKA)(Gen-Probe, Inc., San Diego, Calif.).

However, in view of the myriad of different pathological agents thatmediate disease conditions, a need indeed exists for materials andmethods to collect an accurate clinical sample, e.g. a “snapshot,”representative of a certain gynecological microbiological environment,for example, and maintain the detectability of the diversity ofpathological agents in a single gynecological sample. Methods areparticularly needed for receiving and handling a plurality of singlegynecological swab clinical samples, each having identity and testrequisition information associated therewith, wherein the testrequisition information indicates a test for at least one causativeagent from a plurality of listed agents, and managing informationassociated therewith for reporting a sum of diagnostic results for eachsample.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods and materials for thecollection and maintenance of detectability of a plurality of species ofmicrobiological agents indicative of a gynecological disorder selectedfrom the group consisting of bacteria, fungi, viruses and protozoa, in asingle gynecological sample comprising providing transport media in aresealable container, a sterile swab, and instructions for preparationand handling of a gynecological sample, and an indication of thedetectability of the plurality of species. The current invention isparticularly directed to receiving a plurality of these gynecologicalswab samples and managing material and information associated with thesamples for reporting a sum of diagnostic results for each sample.

Embodiments of the present invention are preferred wherein at least onespecies within the plurality of species is indicative of at least onegynecological disorder, e.g., wherein at least one species within theplurality of species is selected from the group consisting ofBacteroides fragilis, Candida albicans, Candida glabrata, Candidaparapsilosis, Candida tropicalis, Chlamydia trachomatis, Gardnerellavaginalis, Haemophilis ducreyi, Herpes simplex virus subtype 1 (HSV-1),Herpes simplex virus subtype 2 (HSV-2), Human papillomavirus (HPV),Mobiluncus mulieris, Mobiluncus curtisii, Molluscum contagiosum Virus,Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae,Treponema pallidum, Trichomonas vaginalis, Ureaplasma urealyticum, andStreptococcus agalactiae (Group B Streptococcus). Methods are preferredwherein at least two (2), three (3), four (4), five (5), or six (6)species within the plurality of species are selected from the group.

The present invention is directed to a method of receiving and handlinga plurality of clinical samples and managing information associatedtherewith for generating and reporting any of a plurality of differentdiagnostic results from each sample in a timely manner, particularlywithin about thirty (30) hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an example of a Test Requisition Form.

FIG. 2 displays validation data for Bacteroides fragilis wherein the PCRamplicon is 842 by in which each sample was independently inoculated andextracted in triplicate. Lanes 2-4 represent detectability after storageat room temperature for zero days. Lanes 5-7 represent detectabilityafter storage at room temperature for one day. Lanes 8-10 representdetectability after storage at room temperature for two days. Lanes11-13 represent detectability after storage at room temperature forthree days. Lanes 14-16 represent detectability after storage at roomtemperature for four days. Lanes 17-19 represent detectability afterstorage at room temperature for five days. Lanes 20 and 21 are thepositive and negative controls, respectively.

FIG. 3 displays validation data for Mobiluncus mulieris wherein the PCRamplicon is 1015 by in which each sample was independently inoculatedand extracted in triplicate. Lanes 2-4 represent detectability afterstorage at room temperature for zero days. Lanes 5-7 representdetectability after storage at room temperature for one day. Lanes 8-10represent detectability after storage at room temperature for two days.Lanes 11-13 represent detectability after storage at room temperaturefor three days. Lanes 14-16 represent detectability after storage atroom temperature for four days. Lanes 17-19 represent detectabilityafter storage at room temperature for five days. Lanes 20 and 21 are thepositive and negative controls, respectively.

FIG. 4 displays validation data for Candida albicans.

FIG. 5 displays validation data for Candida glabrata.

FIG. 6 displays validation data for Candida parapsilosis.

FIG. 7 displays validation data for Candida tropicalis.

FIG. 8 displays validation data for Chlamydia trachomatis.

FIG. 9 displays validation data for Gardnerella vaginalis:

FIG. 10 displays validation data for Haemophilis ducreyi.

FIG. 11 displays validation data for HSV-1.

FIG. 12 displays validation data for HSV-2.

FIG. 13 displays validation data for Trichomonas vaginalis.

FIG. 14 displays validation data for Ureaplasma urealyticum.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs.

All publications and patents referred to herein are incorporated byreference.

Physicians are generally faced with observing patients' symptoms,obtaining biological samples, and ordering clinical diagnostic tests todetermine the identity of causative agents which mediate pathologicalconditions. Since methods of treatment of pathophysiological conditionsare intimately related to the identity of the causative agent(s) of thecondition, rapid and accurate identification and reporting of thecausative agent(s) is of paramount importance to the practice ofmedicine today. The present invention enables the accurate and rapidreporting of the detection of any of a plurality of biological agentsfrom each clinical sample.

It is an object of the present invention to provide methods andmaterials for the collection and maintenance of detectability of aplurality of species of microbiological agents indicative of agynecological disorder in a single gynecological sample comprisingproviding transport media in a resealable container, a sterile swab,instructions for preparation and handling of a gynecological sample, andan indication of the detectability of a plurality of species. It is alsoan object of the present invention to integrate a method of handling aplurality of these samples and managing information associated therewithfor reporting a sum of diagnostic results for each sample.

The present invention is fundamentally a method for providing certainmaterials for the collection and maintenance of detectability of aplurality of species of microbiological agents indicative of agynecological disorder in a single gynecological sample and receivingand handling a plurality of the clinical samples resulting therefrom andmanaging material and information associated therewith for generatingand reporting a sum of diagnostic results for each sample.

The flow of information and reporting of results from a clinicallaboratory is a fundamental aspect of the present invention. The presentinvention enables the accurate and rapid reporting of the detection ofany of a plurality of biological agents from each clinical sample. Thepresent invention is also a method for receiving and handling aplurality of clinical samples and managing information associatedtherewith to effect rapid diagnostic testing for any of a plurality ofagents in each sample. Although the invention defined by the claimsappended hereto are not necessarily so limited, preferred methods of theinvention are for handling a plurality of single gynecological swabs(e.g., each from a different patient) and managing informationassociated therewith.

A basic embodiment of the method of the present invention involves arapid method of handling a plurality of single gynecological swabsamples and managing information associated therewith for reporting anyof a plurality of different diagnostic results for each sample withinabout fifty (50) hours of receiving the sample (preferably within fortyeight (48) hours, more preferably within about thirty (30) hours, mostpreferably within about twenty four (24) hours). Single gynecologicalswab samples each have identity and test requisition informationassociated therewith, wherein the test requisition information indicatesa test for at least one causative agent, from a choice of a plurality ofagents (for example, between about 5 and about 25 differentmicrobiological agents). The term “causative agent” as used hereinrefers to biological entities that mediate disease conditions,including, but not limited to, microorganisms, e.g., bacteria, fungi,and viruses. Preferred agents, referred to herein as causative agents,include but are not limited to microbiological species associated withpathological gynecological conditions, for example, collected in asingle swab specimen (clinical sample). Causative agents referred toherein include, but are not limited to Bacteroides fragilis, Candidaalbicans, Candida glabrata, Candida parapsilosis, Candida tropicalis,Chlamydia trachomatis, Gardnerella vaginalis, Haemophilis ducreyi,Herpes simplex virus subtype 1 (HSV-1), Herpes simplex virus subtype 2(HSV-2), Human papillomavirus (HPV), Mobiluncus mulieris, Mobiluncuscurtisii, Molluscum contagiosum Virus, Mycoplasma genitalium, Mycoplasmahominis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonasvaginalis, Ureaplasma urealyticum, and Streptococcus agalactiae (Group BStreptococcus), for example.

Proper and timely identification of causative agent(s) of vaginosis, amassive microecologic alteration of the vaginal flora, is a continuousproblem for obstetrician-gynecologists. Bacterial vaginosis (BV), forexample, is related to considerable andpreventable infectious morbidityin non-pregnant women. Bacterial vaginosis mediates endometritis, pelvicinflammatory disease, post-surgical abortion infections,post-hysterectomy infections, an increased risk of HIV acquisition andcervical intraepithelial neoplasia. The diagnosis of BV is complicatedby the polymicrobial nature of the condition. Bacterial Vaginosis (BV),for example, is characterized by a logarithmically increasedconcentration of Gardnerella vaginalis and logarithmically increasedconcentrations of pathogenic bacteria, including Bacteroides spp.,Mobiluncus spp., along with Ureaplasma urealyticum and Mycoplasma spp.Bacteroides fragilis is an anaerobic bacterium that is commonlyassociated with BV. Mobiluncus species, e.g., Mobiluncus mulieris andMobiluncus curtisii, are anaerobic bacteria that are commonly associatedwith BV. Fifty percent of patients diagnosed with BV, however, displayno symptoms.

Mycoplasmas are small (0.2-0.3 nm) membrane bound organisms capable ofindependent self-replication. The most prevalent strains recoverablefrom the genital tract are Ureaplasma urealyticum, Mycoplasma hominisand Mycoplasma genitalium. In some pregnant women, Ureaplasma infectionsare considered to be the cause of chorioamnionitis and prematuredelivery. They are frequently transmitted from mothers to their infants,which may cause a variety of disorders including pneumonia, persistentpulmonary hypertension, and chronic infection of the central nervoussystem. M hominis is associated with pyelonephritis, pelvic inflammatorydisease (PD), spontaneous abortion, and postpartum septicemia and fever.M genitalium has been associated with non-gonoccocal urethritis, acuteendometritis, cervicitis, and pelvic inflammatory disease (PD). Infantsbecome colonized with genital mycoplasmas during birth. Genitalmycoplasma infections are usually diagnosed by culture. However, due toits fastidious slow-growing nature, M genitalium, for example, may takeup to eight (8) weeks to culture.

Candida Vaginitis (CV) is currently the second most common cause ofvaginal infections, with bacterial vaginosis the most common diagnosticentity. However, CV is misdiagnosed in as much as 50% of all cases. Moststudies indicate that CV is a frequent diagnosis among young women,affecting as many as 15% to 30% of symptomatic women visiting aclinician. Candida albicans is one of the major causes of CandidaVaginitis (CV). The widespread use of topical antifungals appears tocontribute to selection for non-albicans yeasts, e.g., C. glabrataaccounts for 7% of all vaginal fungal infections and about 10% ofvaginal yeast isolates. Candida tropicalis is isolated from 1% to 5% ofvaginal yeast isolates and may be associated with a higher rate ofrecurrence after standard treatment. C. parapsilosis accounts for 1% ofvaginal yeast isolates.

Chlamydia trachomatis is the causative agent of a variety of diseasesincluding trachoma and urogenital infections. It is the most commonsexually transmitted bacterial agent and in women it causes cervicitis,urethritis, endometritis and salpingitis. In more complicated cases itmay result in tubal scarring, infertility, and ectopic pregnancy. In menit causes urethritis and proctatitis. Other forms of infection also seenare trachoma, the most preventable form of blindness, and conjunctivitisin neonates.

Neisseria gonorrhoeae is the causative agent of the sexually transmitteddisease gonorrhea. It is the most frequently reported communicabledisease in the United States. In women, the most common presentation isendocervical infection. If left untreated it may develop intovulvovaginitis, salpingitis, and pelvic inflammatory disease (PD).Infections in men range from uncomplicated lower genital tractinvolvement such as urethritis, to the more serious epididymitis,prostatitis, and urethral stricture. Untreated asymptomatic infectionsmay, in certain instances, develop into disseminated gonococcalinfection (DGI).

Genitourinary tract infections due to C. trachomatis and N. gonorrhoeaeare a major cause of morbidity in sexually active individuals. In malesthey may cause epididymitis and urethritis. In females, they can causepelvic inflammatory disease (PID), ectopic pregnancy, and infertility.If left untreated, N. gonorrhoeae may develop into a disseminatedgonococcal infection (DGI). Coinfection with C. trachomatis and N.gonorrhoeae is not uncommon. In fact, up to half of patients diagnosedwith infection of one of these pathogens may be infected with the otherand, therefore, it is important to test all sexually active individualsfor both. Both sexually transmissible pathogens are detectable byswabbing the urethra and/or the cervix (for women) and performing eithera culture and/or a nucleic acid amplification assay (see Van Doornum etal., 2001, Journal of Clinical Microbiology 39(3):829-835).

Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalisare the major causes of leukorrhea. Neisseria gonorrhoeae is thecausative agent of the sexually transmitted disease gonorrhea. In women,the most common symptom of N. gonorrhoeae infection is endocervicalinfection, and if left untreated, may develop into vulvovaginitis andpelvic inflammatory disease. As a protozoan parasite, Trichomonasvaginalis is the causative agent of the sexually transmitted diseasetrichomoniasis. T vaginalis infection is the primary cause of vaginitis,cervicitis and urethritis in women. Routine clinical diagnosis usuallydepends on microscopic identification of the parasite in wet mountpreparations, which are only 60% sensitive as compared toculture-positive women.

The three major causes of Genital Ulcer Disease (GUD) are Herpes simplexvirus, Treponema pallidum (syphilis), and Haemophilis ducreyi(chancroid). As treatment options vary, it is medically necessary toidentify the causative agent of GUD. Currently, the diagnosis of GUD isbased primarily on the clinical presentation of the ulcer itself.However, agent-specific diagnosis based solely on the clinicalevaluations are often obscured by multiple and mixed infections. Tpallidum is the causative agent of the sexually transmitted diseasesyphilis. T pallidum is one of the few major bacterial pathogens ofhumans that cannot be cultivated on artificial medium.

Human Papillomavirus (HPV) subtyping is of clinical significance in viewof the growing evidence for the association of Human Papillomavirus(HPV) subtypes (i.e., HPV-16 and HPV-18) with cervical and ovariancancer. Particularly, 90% of individuals with major grade cervicalintraepithelial neoplasia (CIN 2 and C1N 3) and invasive carcinoma ofthe cervix are also infected with HPV-16 or HPV-18. Moreover, as much as10% to 20% of women in the United States have tested positive to HPV byPapanicolaou (Pap) smear. Since Pap smears cannot differentiate amongHPV subtypes, asymptomatic individuals that are HPV positive are beingdisregarded by the clinicians.

Molluscum contagiosum virus (MCV) is a member of the human pox viruseswhich produces small raised papules or lesions with centralumbilications and a white, firm, curd-like core. Infection occurs duringsexual intercourse. MCV is a common infection in the United States andaccounts for approximately 1% of all undiagnosed skin disorders. Manyphysicians find it necessary to differentiate MCV from Humanpapillomavirus (HPV) or Herpes simplex virus (HSV) infections which havegreater mortality and morbidity.

Vaginal Group B Streptococcus (Streptococcus agalactiae) (GBS), the mostcommon cause of life-threatening infection in newborns, is a commoncause of neonatal septicemia, pneumonia and meningitis. One out of everytwenty babies with GBS dies from the infection. In pregnant women, GBScan cause bladder infections, womb infections, and stillbirth. Manyadults are asymptomatic carriers of GBS in the bowel, vagina, bladder orthroat. Diagnosis by traditional cultures may take several days tocomplete. However, once properly diagnosed, GBS can be treated withantibiotics to prevent the spread from mother to baby.

The term “clinical sample,” as used herein, refers to biological samplesknown in the art. “Clinical sample” includes, for example, but is notlimited to a gynecological swab sample. Particularly, the method of thepresent invention provides materials for the collection and maintenanceof a “snapshot” of a gynecological environment for the detectability ofa plurality of species of microbiological agents in a single,gynecological sample. Clinical samples are generally labeled orotherwise clearly associated, e.g., packaged, with I information thatdistinctly identifies the origin, source and/or destination for theresults for each sample. Each sample is associated with an identifier,e.g., a patient's name, date of birth, and/or social security number,for example, or information otherwise provided by the source to indicatethe distinct origin of each sample.

Methods described herein also comprise (1) providing a transport mediain a resealable container, a sterile swab, and instructions forpreparation and handling of a gynecological sample and a writtenindication of the detectability of the plurality of species, e.g., atest requisition form (see FIG. 1), (2) receiving the completedgynecological sample in a package with a completed test requisitionform, and (3) handling a plurality of these clinical samples andmanaging information associated therewith for reporting any of aplurality of different diagnostic results for each sample in a timelymanner. Reporting may be accomplished by means of facsimile to anattending physician who ordered the test(s), for example, or othermeans, electronic or otherwise, e.g., posting on a private-accessinternet web site, including all means that are usual and customary inthe health-care industry.

The present invention is not drawn to methods for the detection,identification or diagnosis, per se, of any particular microbiologicalspecies, or series of species, or disease condition. The tests, per se,however, whatever tests are used, are not relevant to the subject matterof the present invention. In contrast, methods described herein arespecifically directed toward collection and maintenance of detectabilityof a plurality of species of microbiological agents and handling aplurality of clinical samples and managing material and informationassociated therewith. Particularly, methods of the present invention arefor providing a compilation of certain materials, managing samples andinformation related to the samples proximal in time, i.e., before,during, and after, a determination of any of a plurality of differentpossible diagnostic results for each sample and reporting the results.

EXEMPLARY EMBODIMENT

The Applicants describe herein methods and materials to collect andmaintain the detectability of multiple pathogens from a singlegynecological swab. Specimen viability has been validated for up to fivedays, for example, as illustrated herewith. Longer viability, however,e.g., about 5-10 days, is reasonably expected in view of the datapresented herein. No refrigeration is required. The present invention isnot drawn to methods for the detection, identification or diagnosis, perse, of any particular microbiological species, or series of species, ordisease condition. In contrast, methods and articles of manufacture,i.e., kits, of the present invention are particularly provided for thecollection and maintenance of detectability of a plurality of differentmicrobiological species associated with pathological gynecologicalconditions so that a clinical diagnostic service provider, for example,may enable a physician to collect a single swab specimen (clinicalsample) and order any of a plurality of tests on that sample from theclinical diagnostic service provider, for example. The tests, per se,however, whatever tests are used, are not relevant to the subject matterof the present invention. The Applicants indeed present validation dataherewith merely to demonstrate the viability of the species exemplifiedherein using the materials and methods of the present invention. Sincethe species exemplified herein are well characterized in the art andare, as discovered and illustrated by the Applicants, viable under theconditions described herein, they are indeed detectable by an array ofdifferent methods, e.g., nucleic acid amplification, known to those ofskill in the art.

A method is particularly preferred for the collection and maintenance ofdetectability of a plurality of species of microbiological agentsselected from the group consisting of bacteria, fungi, and viruses, in asingle gynecological sample comprising providing transport media in aresealable container, a sterile swab, and instructions for preparationand handling of a gynecological sample and an indication of thedetectability of the plurality of species.

Microbiological agents that are causative or are otherwise associatedwith gynecological disorders are preferred. Since many different speciesof microbiological agents mediate, or are associated with, or areindicative of gynecological disorders, the present invention provides ameans for handling a plurality of clinical gynecological swab samplesand managing information associated therewith in the process leading upto the identification of at least one causative agent in each sample andreporting the results representative of the ambient population ofmicrobiological agents in each sample at the time each sample was taken.Particularly, the method of the present invention enables a “snapshot”of details corresponding to each a single gynecological sample, within aplurality of samples, to be provided in a valuable period of time bymeans of information management. A single gynecological swab sample isgenerally received in transport media, between about 1 ml and about 5ml, for example, in a resealable container along with a test requisitionform.

Transport media for use in methods of the present invention is auniversal transport media in which viability of a plurality oforganisms, e.g., bacteria, fungi, and viruses, can be sustained undernormal conditions without refrigeration for at least 48 hours. Transportmedia for use in methods of the present invention is preferred whereinthe detectability of species selected from the group consisting ofBacteroides fragilis, Candida albicans, Candida glabrata, Candidaparapsilosis, Candida tropicalis, Chlamydia trachomatis, Gardnerellavaginalis, Haemophilis ducreyi, Herpes simplex virus subtype 1 (HSV-1),Herpes simplex virus subtype 2 (HSV-2), Human papillomavirus (HPV),Mobiluncus mulieris, Mobiluncus curtisii, Molluscum contagiosum Virus,Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae,Treponema pallidum, Trichomonas vaginalis, Ureaplasma urealyticum, andStreptococcus agalactiae (Group B Streptococcus) is maintained at roomtemperature for five (5) days.

An example of a general support media for a variety of organisms in aclinical sample is Stuart's medium (see, e.g., Stuart et al., 1954, Theproblem of transport of specimens for culture of Gonococci. CanadianJournal of Public Health. 45(2):73-83). Stuart's medium is a well-knownbuffered transport medium which includes the component sodiumglycerophosphate to permit minimal multiplication and sodiumthioglycollate as a reducing agent to prevent oxidation within thesample. Stuart's medium, however, contains no nutrients. The absence ofnutrients retards the growth of commensal organisms within the samplewhich can multiply and overgrow the less hardy pathogens. However, theabsence of nutrients in Stuart's media can be detrimental to theviability of less hardy pathogens. Preferred transport medium comprisesan aqueous balanced salt solution buffered at approximatelyphysiological pH, at least one protein stabilizer, and combinations ofcarbohydrate and amino acid nutrient sources. The medium is buffered tomaintain physiological pH and may include a pH indicator in order toindicate variation of pH outside the physiological pH range. The mediumfurther comprises antimicrobial and antifungal agents and can includegelatin. Transport media, for example, is liquid media whereindetectability of a plurality of species of microbiological agentsincluding bacteria, fungi, and viruses is maintainable under normalconditions, without refrigeration, for at least 72 hours. Exampletransport media herein is demonstrated to be suitable for maintainingthe detectability, for example, of a plurality of species ofmicrobiological agents including bacteria, fungi, and viruses undernormal conditions, without refrigeration, for about five (5) days (e.g.,UTM-RT Transport Medium, Copan Diagnostics Inc., Corona, Calif.).

Transport medium, for example, consists of modified Hank's balanced saltsolution supplemented with bovine serum albumin, cysteine, gelatin,sucrose, and glutamic acid. The pH is buffered with HEPES buffer, forexample. Phenol red is used to indicate pH. Vancomycin, amphotericin B,and colistin are incorporated in the medium to inhibit growth ofcompeting bacteria and yeast. The medium is isotonic and non-toxic tohuman cells. Example components comprise, for example, Hank's BalancedSalts, Bovine Serum Albumin, L-Cysteine, Gelatin, Sucrose, L-GlutamicAcid, HEPES Buffer, Vancomycin, Amphotericin B, Colistin, and PhenolRed; pH 7.3 +/−0.2 @ 25° C. Antimicrobial compositions can includevancomycin, gentamicin, colistin, or amphotericin B.

A general transport medium of about 1.0 liter (total volume) may beprepared in the following aqueous composition: gelatin, 0-20.0 g; sugar,65-75 g; HEPES, 5-7 g; KCl, 0.3-0.6 g; L-glutamic acid, 0.5-1.0 g;phenol red, 5-15 mg; CaCl₂, 0.1-0.5 g; MgSO₄ 7H₂O, 0.1-0.3 g; bovineserum albumin V, 1.0-20.0 g; vancomycin, 0.01-0.05 g; colistin,100,000-250,000 units; and amphotericin B, 0.5-3.0 mg.

An example may include the following ingredients in about 995 ml ofwater q.s. 1 liter: sucrose, 68.46 g; HEPES, 5.96 g; KCl, 0.4 g;L-glutamic acid, 0.72 g; phenol red, 11.0 mg; CaCl₂, 0.27 g; MgSO₄.7H₂O, 0.20 g; BSA, 5.0 g; gelatin, 5.0 g; vancomycin, 0.025 g; colistin,200,000 units; and amphotericin B, 1.0 mg. The pH of the medium can beadjusted with acidic or basic solutions to arrive at a final pH withinphysiological limits (see, e.g., U.S. Pat. No. 5,702,944, entitledMicrobial Transport Media, which is herein incorporated by reference inits entirety).

Preferred commercially available and validated examples of transportmedium for use in methods and articles of the present invention include,for example, the following: UTM-RT Transport Medium, BD Cellmatics ViralTransport Medium® (Becton, Dickinson & Company, Sparks, Md.), MultitransCulture Collection and Transport System (Starplex Scientific, Etobicoke,Ontario, Canada), The ThinPrep® Pap Test Preservcyt® Solution (CYTYCCorporation, Boxborough, Mass.), SurePath® (Tripath Imaging Inc.,Burlington, N.C.).

Preferred methods of the present invention provide a transport media ina resealable container, a sterile swab, instructions for preparation andhandling of a gynecological sample and a written indication of thedetectability of the plurality of species.

A labeled screw-cap tube is preferred, for example, as a resealablecontainer, which contains a volume of transport medium, between about 1ml and about 5 ml, for example, suitable for accurate collection andmaintenance of a population of microbiological agents representative ofa gynecological environment. A resealable container optionally containsglass beads (three 3 mm beads, for example). Although not required to beprovided, per se, in methods and the specified compilation of materialsdescribed herein, at least one sterile swab, well-known in the art ofgynecology, is preferred in the materials of a packaged kit describedherein for obtaining the gynecological sample. At least one sterile swabfor obtaining the gynecological sample, for example, is preferablysupplied with the transport media in a package along with writteninstructions for preparation and handling of a gynecological sample inthe media, and a written indication of the detectability of a pluralityof species, e.g., a test requisition form. The transport medium may besupplied, however, alone in a package which comprises a writing, i.e., awritten indication of the detectability of a plurality of species, forexample, a test requisition form which lists a plurality of species ofmicrobiological agents described herein for selection, for example, bythe attending physician. The compilation of materials described hereinis preferably provided in methods described herein in a container, i.e.,a package. The package preferably contains the materials in akit-format. Written instructions for preparation and handling of agynecological sample in the media are preferred to be included in thecompilation of materials otherwise described herein as a packaged kitintended, designated, and prepared for the specific purpose ofcollection and maintenance of detectability of a plurality of species ofmicrobiological agents described herein. Once a swab sample iscollected, it should be placed immediately into the transport tube whereit comes into contact with transport medium.

A first set of example instructions are as follows:

-   1. Collect the single specimen with a swab (Polyester (Dacron)    tipped swabs are suitable).-   2. Aseptically remove the cap from the transport media.-   3. Insert swab into the tube with the transport medium.-   4. Break swab shaft by bending it against the tube wall.-   5. Replace cap to tube and close tightly.-   6. Label the tube with appropriate patient information.-   7. Complete the test requisition form included herewith.-   8. Send these items in the pre-addressed postage materials included    herewith to the laboratory for immediate analysis.

A second set of example instructions are as follows:

-   1. Visualize cervix with speculum without lubricant.-   2. Remove mucus and/or secretions from the cervix with a swab,    discard swab.-   3. Firmly, yet gently, sample the endocervical canal with sterile    swab for 10 seconds.-   4. Place the swab into the transport vial.-   5. Be sure the cap is sealed tightly.

Specimen collection swab options include, for example: one regular sizeplastic shaft swab with polyester fiber tip; two regular size plasticshaft swabs with polyester fiber tips; one regular size plastic shaftswab and one Minitip plastic shaft swab pre-scored for easy breakage,both with polyester fiber tips; one Minitip plastic shaft swab withpolyester fiber tip pre-scored for easy breakage; one Combo stainlesssteel wire-plastic shaft Minitip swab with polyester fiber tip: oneregular size plastic shaft swab and one Combo stainless steelwire-plastic shaft Minitip swab, both with polyester fiber tips. Thesedifferent swab applicator shafts facilitate the collection of specimensfrom various sites on a patient.

Receiving a Plurality of Single Gynecological Swab Samples

A “plurality of samples” is an inclusive term which refers to aplurality of single samples from different patients. A “plurality” ofsamples generally refers to a substantial number of biological samplesreceived by a clinical lab within a twenty four (24) hour period, forexample.

A plurality of samples, however, as used herein may refer to as few asseveral samples, e.g., about ten (10), or about five thousand (5,000)samples, for example, to be processed. Each sample has identity and testrequisition information associated therewith, wherein the testrequisition information indicates a test for at least one causativeagent, from a choice of a plurality of agents, from a list of betweenabout 5 and about 25 different microbiological agents, for example. Forthe purpose of illustration of the complexity of information associatedwith a plurality of single gynecological swabs and test requisitioninformation associated therewith, each sample with a test requisitionform which indicates the detectability of twenty (20) different agents,for example, has the possibility of about 400 different diagnosticresults, for that single sample. This, combined with the fact that aplurality of samples (e.g., 500) are received to be processed together,that day, illustrates the complexity of information associated therewithto be managed in order to handle the plurality of clinical samples forreporting any of a plurality of different diagnostic results for eachsample in a timely manner. The current invention is particularlydirected to methods wherein the test requisition information indicates atest for at least one (1), preferably at least two or three (2 or 3),e.g., between 4 and about 6, causative agents from a choice (list) of aplurality of agents. Embodiments of the present invention include, forexample, wherein the plurality of species comprise at least one speciesselected from the group consisting of Molluscum contagiosum Virus,Mycoplasma genitalium, and Mycoplasma hominis.

The information in the system, i.e., the identity of the sample (e.g.,sample identifier or identification tag) and test requisitioninformation, i.e., tests specifically requested to be performed on thatsample, is processed to designate a test on each sample for at least onecausative agent. Accordingly, methods described herein comprise enteringidentity and test requisition information associated with each sampleinto a system to create a requisition file for each sample. The term“system” as used herein refers generally to a system of recording andmanaging information, a computer implemented information managementsystem to manage the flow of information and, in certain embodiments, tocontrol instrumentation, throughout the process of the presentinvention. This system is preferred, but, however, is not required. Acomputer is generally employed to receive the identity and testrequisition information associated with each sample. The information maybe entered manually into a server, for example, to create a testrequisition file for each sample which comprises the sample informationand the test requisition information. A listing, file for example, ofthe identity of all samples for each test is created. If twentydifferent tests are to be performed (for twenty different pathologicalagents), for example, twenty different lists of sample identifiers arecreated. If a certain sample will be subject to three different tests,for example, that sample identifier will be on at least three separatelists corresponding to those three different tests. In some embodimentsof the present invention a computer implemented system performscalculations and/or controls instrumentation.

A method for the collection and maintenance of detectability of aplurality of species of microbiological agents selected from the groupconsisting of bacteria, fungi, and viruses, in a single clinical sampleand for handling a plurality of samples and managing informationassociated therewith for reporting a sum of diagnostic results for eachsample is preferred which comprises providing transport media in aresealable container with instructions for preparation and handling of asample and an indication of the detectability of the plurality ofspecies, receiving a plurality of samples, each having identity and testrequisition information associated therewith wherein the testrequisition information indicates a test for at least one species from aplurality of species, entering the information into a system to create arequisition file for each sample, processing the information to create alist of samples to be tested for each species, dispensing an aliquotcorresponding to each sample into an individual vessel, to create asecondary sample, for each designated test for different species on eachsample, assembling a general supply of master reagent mix for eachdifferent test, combining an aliquot of master reagent mix for each testwith each corresponding secondary sample to produce a diagnostic testreaction for each secondary sample, determining the presence or absenceof a certain product of each reaction to produce a result, recording theresult of each reaction, combining the result of each reaction derivedfrom each primary sample into the requisition file for each sample onthe system, thereby producing a sum of results for each sample, andreporting the results. Methods are particularly preferred wherein atleast one species within the plurality of species is indicative of atleast one gynecological disorder. Methods of the present invention arepreferred wherein the plurality of species comprises Chlamydiatrachomatis and Neisseria gonorrhoeae. Methods of the present inventionare preferred wherein the plurality of species comprises Gardnerellavaginalis, Mobiluncus mulieris, Mobiluncus curtisii, and Bacteroidesfragilis. Methods of the present invention are preferred wherein theplurality of species comprises Candida albicans, Candida glabrata,Candida parapsilosis, and Candida tropicalis. Methods of the presentinvention are preferred wherein the plurality of species comprisesMycoplasma genitalium, Mycoplasma hominis, and Ureaplasma urealyticum.Methods of the present invention are preferred wherein the plurality ofspecies comprises Herpes simplex virus, Treponema pallidum, andHaemophilis ducreyi. Methods of the present invention are preferredwherein at least one (1) species of the plurality of species is selectedfrom the group consisting of Molluscum contagiosum Virus, Mycoplasmagenitalium, and Mycoplasma hominis.

Nucleic Acid is Extracted from Each Sample

Established procedures for DNA extraction are used (see Example II). Inbrief, swabs are thoroughly mixed in the transport media containedwithin the transport vials. 470 μI of transport media is mixed with 25μI of 10% sodium dodecyl sulfate, and 12 μI of freshly preparedDNase-free proteinase-K (10 mg/ml), then incubated for 2 hours at 55° C.DNA is phenol:chloroform:isoamyl alcohol extracted and recovered byethanol precipitation. DNA is pelleted, dried in a speed vacuum, andresuspended in 20 μ1 TE buffer. DNA concentration is calculated byabsorbance 260/280 readings and was adjusted to 0.2 μg/μ1 prior to PCRanalysis. Quantitation, however, is preferred using a fluorometer.Quantitation, however, is preferred using a fluorometer such as oneavailable from Turner BioSystems, Inc., Sunnyvale, Calif.

The nucleic acid from each sample is diluted to about 200 ng/μ1, forexample, with water, for example, to provide a standardized primarynucleic acid solution corresponding to each sample. An aliquot ofnucleic acid from each sample is dispensed into a separate individualvessel to create a secondary sample corresponding to each designatedtest on each sample. A general supply of master reagent mix, e.g.,real-time PCR mix, for each test for each different causative agent isprepared. An aliquot of each master reagent mix is combined with each,corresponding secondary nucleic acid sample for each test to produce adiagnostic test reaction for each secondary sample. Each reaction isincubated and preferably monitored in real-time.

The presence or absence of a certain product of each reaction to producea result is determined. The result of each reaction is recorded in thesystem. The result of each reaction derived from each primary solutionis combined into the requisition file for each sample on the system,thereby identifying at least one causative agent in each sample, and theresults of the identification are reported.

A rapid method of handling a plurality of clinical samples and managinginformation associated therewith for identifying at least one causativeagent in each sample and reporting results comprises receiving aplurality of samples, each having identity and test requisitioninformation associated therewith wherein the test requisitioninformation indicates a test for at least one causative agent, enteringthe information into a system to create a requisition file for eachsample, processing the information to designate a test on each samplefor at least one causative agent, dispensing an aliquot corresponding toeach sample into an individual vessel to create a secondary sample foreach designated test, assembling a general supply of master reagent mixfor each test for a different causative agent, combining an aliquot ofeach master reagent mix with each corresponding secondary sample foreach test to produce a diagnostic test reaction for each secondarysample, incubating each reaction, determining the presence or absence ofa certain product of each reaction to produce a result, recording theresult of each reaction, combining the result of each reaction derivedfrom each primary sample into the requisition file for each sample onthe system, thereby identifying at least one causative agent in eachsample, and reporting the results of the identification.

A preferred method of handling a plurality of clinical samples andmanaging information associated therewith for identifying at least onecausative agent in each sample and reporting results comprises receivinga plurality of samples, each having identity and test requisitioninformation associated therewith wherein the test requisitioninformation indicates a test for at least one causative agent, enteringthe information into a system to create a requisition file for eachsample, extracting nucleic acid from each sample, quantitating thenucleic acid, diluting the nucleic acid from each sample to provide astandardized primary nucleic acid solution corresponding to each sample,processing the information in the system to designate a real-time PCRtest on each nucleic acid for at least one causative agent, dispensingan aliquot of the primary solution from each sample into a separateindividual vessel to create a standardized secondary nucleic acid samplefor each designated test on each sample, assembling a general supply ofmaster reagent mix for each test for a different causative agent,combining an aliquot of each master reagent mix with each correspondingsecondary nucleic acid sample for each test to produce a diagnostic testreaction for each secondary sample, incubating each reaction,determining the presence or absence of a certain product of eachreaction to produce a result, electronically recording the result ofeach reaction, in the system, combining the result of each reactionderived from each primary solution into the requisition file for eachsample on the system, thereby identifying at least one causative agentin each sample, and reporting the results of the identification.Preferred methods of the invention described herein employ quantitatingnucleic acid from each sample by means of a fluorometer. Methods of thepresent invention preferably comprise generation of diagnostic resultsby means of real-time PCR.

To Maximize the Success of Clinical Diagnostic Methods Described Herein

A clinical diagnostic laboratory should be physically set up so thatspecimen separation and extractions occur in a separate room, using a“Class II Biohazard Safety Hood.” PCR preparation should occur in aseparate room, within one of many PCR cabinets which are dedicatedsolely to PCR preparation. The PCR amplification should occur inthermocyclers located in an enclosed room. For post-amplificationprocess of conventional PCR reactions, gel electrophoresis should beperformed in yet another physically separate room. UV lights should beused in the PCR hoods and commercial solvents, such as DNAway (MolecularBio Products, San Diego, Calif.), to decontaminate all work surfacesprior to and at the completion of any procedures occurring in that area.Sterile, disposable plasticware should be used wherever possible. Allglassware should be autoclaved. All PCR reactions should be performed inindividual closed tube systems as opposed to 96-well microtiter platesto eliminate cross contamination. Real-time PCR assays, for example, donot require gel electrophoresis and therefore eliminatepost-amplification specimen handling. All technicians should onlymanipulate one specimen at a time. This means when a reagent is added toa batch of specimens, it occurs one tube at a time. The next patient'sreaction tube is not opened until the previous patient's tube has beenclosed. Pipette tips used when dispensing reagents should be filtered toprevent aerosol contamination and are also replaced between allspecimens. Reagents used during PCR preparation may be aliquoted into1.5 ml microcentrifuge tubes, for example, as opposed to dispensationinto stock bottles of greater·volumes. This enables the laboratory tomonitor potential contamination closely and discard any reagents, ifever necessary. The use of separate rooms is recommended todecontaminate an entire room if contamination is suspected. Positive andnegative controls should be employed to assess false positives as wellas false negatives. Uracil-N-glycosylase is recommended in everyreaction to minimize, if not eliminate, any possible carry-overcontamination.

Primers

Any pair of PCR primers may be employed in methods of the presentinvention that function to amplify target nucleic acids. The art ofselection and synthesis of PCR primers in order to amplify a particulartarget sequence is indeed well-known to those of ordinary skill in theart. Typically, oligonucleotide primers are about 8 to about 50nucleotides in length. Primers 12 to 24 nucleotides in length arepreferred. Primer pairs that amplify particular nucleic acid moleculescan be designed using, for example, a computer program such as OLIGO(Molecular Biology Insights, Inc., Cascade, Col.). A biotin moiety, forexample, is preferably attached to the 5′ end of one of the primers tofacilitate sample preparation for “pyrosequencing,” a term which denotesthe nucleotide sequencing method described in U.S. Pat. Nos. 6,210,891and 6,258,568; Ronaghi et al., 1998, A sequencing method based onreal-time pyrophosphate. Science 281:363-365; and Ronaghi, 2001,Pyrosequencing sheds light on DNA sequencing. Genome Research 11:3-11.Other entities, however, well known to those of skill in the art, maysimilarly be incorporated, integrated, or attached to one of the primersto facilitate the isolation of the resulting amplicon forpyrosequencing.

Real-Time PCR

Quantitative real-time PCR is a preferred method of amplification of atarget nucleic acid. Products used to accomplish the methods arecommercially available from several manufacturers including, but notlimited, to Corbett Research (Mortlake, Australia), Cepheid (Sunnyvale,Calif.), BioRad (Hercules, Calif.), and Applied Biosystems (Foster City,Calif.). The Corbett Research (Melbourne, Australia) Rotor-Gene′″3000,for example, is a centrifugal, real-time DNA amplification system.

Validation studies exemplified herein are merely a general demonstrationof the utility and value of the present invention, namely a method forthe collection and maintenance of detectability of a plurality ofspecies of microbiological agents in a single gynecological sample, inthe grand scheme of clinical diagnostics. The legitimacy of the PCRmethod is not a relevant factor, as its utility as an invaluablemolecular biological tool has already been well established in theinternational scientific literature through the publication of thousandsof peer-reviewed articles. Particularly, molecular amplification ofnucleic acids by means of PCR is well-known to those of ordinary skillin the art, i.e., the ability of the PCR method to detect geneticsequences specific to a target pathogen within a given clinicalspecimen. The Applicants particularly highlight, however, that themethods described herein, which comprise providing transport media in aresealable container to a physician, clinical lab, or medicalinstitution, with instructions for preparing and handling agynecological sample, along with a test requisition form which indicatesthe detectability of a plurality of species described herein, affect theability of a physician, for example, to collect a single swab sample ofa gynecological environment for the maintenance of detectability of aplurality of species of microbiological agents. Example assays designedto test sensitivity, specificity, interference and optimization wereperformed to validate the operability of the methods and materialsdescribed herein, as claimed. In other words, PCR methods or reagentsemployed to detect microbiological agents are not relevant to the scopeof the subject matter of the claims appended hereto. In contrast, thepresent invention is solely drawn toward methods and certain materialsfor collection and maintenance of detectability of a plurality ofspecies of microbiological agents in a single gynecological swab sample.

Sensitivity refers to a method's ability to detect very minute amountsof a substance or organism. The frequency of a positive test result inpatients who have the disease the test is designed to detect, isexpressed mathematically as follows:

${Sensitivity} = \frac{{True}\mspace{14mu} {Positives} \times 100}{{{True}\mspace{14mu} {Positives}} + {{False}\mspace{14mu} {Negatives}}}$

Sensitivity studies were initially performed by purchasingwell-characterized, validated organisms from the American Tissue CultureCollection (ATCC, Manassas, Va.). The DNA of the virus, bacteria, orfungi is then extracted and quantitated. Standards of knownconcentrations are used to determine the assay's ability to detectvarying concentrations of genetic material. The extracted DNA isserially diluted to concentrations of 1:10, 1:100, 1:1,000 and 1:10,000.By evaluating the presence of bands in these dilutions of knownconcentrations, the sensitivity of a particular test can be established.For real-time PCR assays, the fluorescence acquisition profile generatedfrom the amplification of the serial dilutions is analyzed. A regionencompassing the genetic target of the assay is generally subcloned intoa vector system. Through quantitation of the vector and the optimizationof the assay as described infra, as few as 10 genomic equivalents of thepathogen can be reproducibly detected.

Specificity studies were used to assess the quality of the primerselection for the assay by determining if their organisms' DNA willcross-react in any way leading to false positives. Initially as atheoretical test, primers are cross-referenced against the billions ofother genetic sequences which have been deposited in the publicdatabases by international researchers and any potential conflicts areavoided. Next as an experimental confirmation, the primers and probesare assayed for their inability to amplify dozens of other knownbacterial, viral, and fungal organisms which have been identified ashuman pathogens. An aliquot of the characterized positive control isalso spiked in a suspension consisting of the DNA of numerous otherorganisms to ensure that the particular pathogen target genome is in noway masked or inhibited by other genomic sequences.

Interference studies are used to determine if other substances inherentto the specimen type will interfere with detection by PCR. Certaineffects, such as masking the organism's target to produce a falsenegative, or cross-reactivity to produce a false positive are analyzed.Characteristics, such as the microcosm of normal flora of the genitaltract, the abundance of various proteins found in blood, and naturalinhibitors commonly found in other body fluids, such as urine, can allhave detrimental effects on the PCR process, unless accounted for duringthe initial processing and extraction procedures.

Optimization studies are the final step of the validation process. Inthese assays, the concentrations of various reagents are varied such astemplate DNA, MgCl₂, and primers, and probes as well as the temperatureand duration of each step of the thermocycling parameters to improve theclarity of bands or the intensity of signals, as well as eliminatestreaks, multiple banding, or haziness, which can impede thevisualization of the PCR products or interpretation of real-time PCRresults. A method is preferred wherein a progress of at least onereaction is optically monitored by means of the system and/or whereinthe presence or absence of a product of at least one reaction isoptically determined and electronically recorded by the system.

ADDITIONAL REFERENCES

-   1. Adelson et al., 2005, Simultaneous detection of herpes simplex    virus types 1 and 2 by real-time PCR and pyrosequencing. Journal of    Clinical Virology 33:25-34. (manuscript published online on Nov. 14,    2004).

2. Trama et al., 2005, Detection of Candida species in vaginal samplesin a clinical·laboratory setting. Infectious Diseases in Obstetrics andGynecology 13(2):63-67.

3. Trama et al., 2005, Detection and identification of Candida speciesassociated with Candida vaginitis by real-time PCR and pyrosequencing.Molecular and Cellular Probes 19(2): 145-152.

4. Trama et al. Analyzing Candida albicans gene mutations thatcontribute to azole resistance by pyrosequencing. American College ofObstetricians and Gynecologists 52nd Annual Clinical Meeting, May 1-5,2004, Philadelphia, Pa.

5. Trama et al. Novel technique for identification of vulvovaginalcandidiasis by real-time PCR and pyrosequencing. American College ofObstetricians and Gynecologists 52′1 Annual Clinical Meeting, May 1-5,2004, Philadelphia, Pa.

6. Adelson et al., Diagnosis of Neisseria gonorrhea, Chlamydiatrachomatis, and Trichomonas vaginalis by real-time PCR. AmericanCollege of Obstetricians and Gynecologists 52nd Annual Clinical Meeting,May 1-5, 2004, Philadelphia, Pa.

7. Mordechai et al., Prevalency of Candida species associated withCandida vaginitis in the United States. American Society of Microbiology104th General Meeting, May 23-27, 2004, New Orleans, La., Poster C-108.

8. Adelson et al., Development of a real-time PCR assay for thesimultaneous detection of herpes simplex virus types 1 and 2 withconfirmation by pyrosequencing technology. American Society ofMicrobiology 104th General Meeting, May 23-27, 2004, New Orleans, La.,Poster C-273.

9. Naurath et al., Detection and quantification of Gardnerella vaginalisby real-time PCR. American College of Obstetricians and Gynecologists53rd Annual Clinical Meeting. May 7-11, 2005, San Francisco, Calif.

10. Trama et al, Detection of molluscum contagiosum virus by real-timePCR and pyrosequencing. American Society of Microbiology 105th GeneralMeeting, June 5-9, 2005, Atlanta, Ga.

11. Feola et al., Detection of Ureaplasma urealyticum, Mycoplasmahominis, an Mycoplasma genitalium by real-time PCR and pyrosequencing.American Society of Microbiology 105th General Meeting, June 5-9, 2005,Atlanta, Ga.

12. Gygax et al., Erythromycin and clindamycin resistance in Group BStreptococcal clinical isolates. Presented by Dr. Martin E. Adelson atthe 45th ICAAC (Interscience Conference on Antimicrobial Agents andChemotherapy) Meeting in Washington DC on Dec. 16, 2005.

13. Adelson et al., Evaluation of UTM-RT for the molecular detection ofa plurality of OB/GYN related pathogens. Presented by Dr. Martin E.Adelson at the 45th ICAAC (Interscience Conference on AntimicrobialAgents and Chemotherapy) Meeting in Washington D.C. on Dec. 17, 2005.

EXAMPLES

Example I

Validation Studies

To determine if Copan UTM-RT media is suitable for the molecularamplifications diagnostic testing, the following pathogens werepurchased from ATCC and detection assays were performed:

TABLE 1 Pathogen ATCC Catalogue Number 1 Bacteroides fragilis 23745 2Candida albicans 18804 3 Candida glabrata  2001 4 Candida parapsilosis10233 5 Candida tropicalis 13803 6 Chlamydia trachomatis VR-901B 7Gardnerella vaginalis 14018 8 Haemophilis ducreyi 27721 9 Herpes SimplesVirus-1 VR-1544 10 Herpes Simples Virus-2 VR-734 11 Mobiluncus mulieris35243 12 Mycoplasma hominis 14027 13 Neisseria gonorrhoeae 27628 14Trichomonas vaginalis 30246 15 Ureaplasma urealyticum 27618

Simulation of a Positive Clinical Specimen

Pathogens were purchased from ATCC in a lyophilized pellet form. Eachpellet was dissolved in five ml of TE-buffer (10 mM Tris, pH 7.5, and 1mM EDTA) in case of bacteria or yeast liquid media (10 g of yeastextract, 20.g of peptone dissolved in 1 L of distilled water, pH 7) incase of fungi. Virus cultures were purchased from ATCC as two ml liquidcultures. Dilutions were subsequently prepared as follows:

TABLE 2 Concentration (Designation) 1:1 (A) 1:10 (B) 1:100 (C) OriginalResuspension 600 μ1  60 μI  6 Ill TE Buffer (Bacteria, Virus) or  0 μI540 μI 594 μI Yeast Liquid Media (Fungi)

DNA was extracted from 500 μI of A, B, and C dilutions using standardlaboratory phenollchloroform/ethanol precipitation protocols. Forpositive controls, pathogen-positive clinical specimens were identifiedfrom the initial laboratory diagnostic tests and 500 μI of thecorresponding original cervical swab media specimen was extracted.Previously validated real-time PCRs for each set of pathogens wasperformed on DNA extracted from Dilutions A, B, and C as well as theclinical samples. Rotor-Gene software calculated CT values for the threeATCC dilutions and the clinical specimens (Rotor-Gene 3000 instrument).The CT values of the dilutions were compared with that obtained for theclinical specimens and a “simulated dilution” was extrapolated for thesubsequent studies of the Copan UTM-RT transport medium. Based uponthese studies, the following was selected:

TABLE 3 TE Buffer ATCC (Bacteria, Resuspension Virus) or Yeast OverallATCC used in this Liquid Media Dilution Resuspension experiment (Fungi)of pellet Bacteria 5 ml TE buffer 5 μI 245 μI 1:250 added to pelletFungi 5 ml TE buffer 5 μI 245 μI 1:250 added to pellet Viruses 2 mlculture from 2 μI 198 μI 1:100 ATCC

The Applicants' studies suggest that spiking an ATCC culture (pelletsuspended in 5 ml of medium or buffer) diluted at 1:50 simulates theconcentration of bacterial and fungal pathogens (i.e., 250-fold dilutionof ATCC culture) and 1:100 dilution simulates the viral pathogen(100-fold dilution of ATCC culture) in the clinical sample.

Studying the Stability of the Pathogen

For validation studies, Copan UTM-RT transport medium (Lot # A 303CS02)as provided by the manufacturer was pooled in a sterile bottle. Basedupon the simulated dilutions described above for each pathogen, thefollowing cocktails were prepared:

TABLE 4 Per vial (A, B, & C) μ1 Copan Cocktail Pathogens μI Pathogen*UTM-RT 1 Candida albicans 80 μ1 3840 μ1 Neisseria gonorrhoeae 80 μI 2Candida parapsilosis 80 μ1 3800 μ1 Chlamydia trachomatis 80 μI HerpesSimplex Virus-1 40 μI 3 Candida glabrata 80 μI 3800 μ1 Herpes SimplexVirus-2 40 μ1 Trichomonas vaginalis 80 μI 4 Candida tropicalis 80 μ13760 μ1 Mobiluncus mulieris 80 μI Ureaplasma urealyticum 80 μI 5Bacteriodes fragilis 80 μI 3840 μ1 Mycoplasma hominis 80 μI 6Gardnerella vaginalis 80 μI 38400 Haemophilis ducreyi 80 μI *Dilutionprepared for each pathogen as detailed in Table 4.

Each cocktail was prepared in triplicate (15 ml tubes) and designated A,B, or C. Pathogen culture solution was added to obtain desiredconcentration which mimics the pathogen load in a positive clinicalsample (1:250-fold dilution for ATCC bacterial and fungal culture and1:100-fold dilution for ATCC virus culture). 500 μl of the above mix wastransferred to three separate microcentrifuge tubes labeled Day 0 to 5.

Inoculated media vials of each cocktail were incubated at roomtemperature. At 24 hour intervals starting with Day 0 through Day 5,three microcentrifuge tubes were transferred to −20° C. storage.Aliquots from each vial were extracted for DNA by standard laboratoryprocedures after Day 5. Conventional and real-time PCR reactions foreach pathogen on the appropriate cocktail followed. The summary ofresults is as follows:

TABLE 5 # Positive Time Pts./ Pathogen Type of PCR # Specimens Tested 1Bacteriodes fragilis Conventional PCR 18/18 (see FIG. 2) 2 Candidaalbicans Real-time PCR 18/18 (see FIG. 4) 3 Candida glabrata Real-timePCR 14/18 (see FIG. 5) 4 Candida parapsilosis Real-time PCR 18/18 (seeFIG. 6) 5 Candida tropicalis Real-time PCR 18/18 (see FIG. 7) 6Chlamydia trachomatis Real-time PCR 18/18 (see FIG. 8) 7 Gardnerellavaginalis Real-time PCR 18/18 (see FIG. 9) 8 Haemophilis ducreyiReal-time PCR 18/18 (see FIG. 10) 9 Herpes Simples Virus-1 Real-time PCR18/18 (see FIG. 11) 10 Herpes Simples Virus-2 Real-time PCR 18/18 (seeFIG. 12) 11 Mobiluncus mulieris Conventional PCR 18/18 (see FIG. 3) 12Mycoplasma hominis Conventional PCR  6/6 13 Neisseria gonorrhoeaeReal-time PCR 18/18 14 Trichomonas vaginalis Real-time PCR 18/18 (seeFIG. 13) 15 Ureaplasma urealyticum Real-time PCR 18/18 (see FIG. 14)

Example II

DNA Extraction from Transport Media

For DNA extraction, see, e.g., Goessens et al., 1995, Influence ofvolume of sample processed on detection of Chlamydia trachomatis inurogenital samples by PCR. Journal of Clinical Microbiology 33:251-253.

The following steps outline the procedure to isolate and purify DNA fromtransport media. The specimen is submitted as a self-contained unit withtransport media.

Proteinase K: 100 μl Tris (pH 7.5), 4.9 ml ddH₂O, 5 ml Glycerol.Dissolve well and store at −20° C. as 500 μl aliquots.

10% SDS: 10 g SDS in 100 ml of ddH₂O.

Equipment:

-   Disposable pipette tips-   Disposable transfer pipette-   Laboratory timer-   1.5 ml microcentrifuge tube-   55° C. water bath-   Pipettes to deliver a range of 1-1000 μl

Procedure:

-   1. Mix the swab thoroughly in the transport media.-   2. Pipette 470 μl of transport media into a labeled microcentrifuge    tube.-   3. Add 25 μl of 10% SDS and 12 Al of Proteinase K. Mix well.-   4. Incubate for 2 hours in 55° C. water bath.-   5. After 2 hours, place 200 μl of Tris saturated phenol and 200 μl    of chlorofonn:isoamyl alcohol (24:1) in the tube. Shake the tube to    mix the layers.-   6. Centrifuge at 14,000 rpm for 5 minutes at room temperature. This    will separate the layers.-   7. Remove the top chloroform layer (containing the DNA) being    careful not to pipette any of the bottom or middle layers. Place    this into another labeled microcentrifuge tube. The first tube    containing the remaining layers may be discarded.-   8. To this new tube add 0.1 × volume of 3 M sodium acetate. Also add    2× volumes of cold 100% ethanol Vortex and place in −20° C.    overnight.-   9. Centrifuge the tube at 14,000 rpm at 4° C. for 10 minutes. This    will pellet the precipitated 20 DNA.-   10. Remove and discard the supernatant. Add 1000 μl of 70% ethanol    to wash the pellet. Slightly resuspend the pellet.-   11. Centrifuge the tube again at 14,000 rpm at 4° C. for 5 minutes    to form a pellet.-   12. Place the tube with the top open into the CentriVap (Labconco,    Kansas City, Mo.). Spin at 35° C. for approximately 15 minutes. Spin    until the pellet is dry, being very careful not to overdry.-   13. Resuspend the pellet in 20 Al of ddH₂O.-   14. Quantitate the DNA using a spectrophotometer.

All publications and patents referred to herein are incorporated byreference. Various modifications and variations of the described subjectmatter will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tothese embodiments. Indeed, various modifications for carrying out theinvention are obvious to those skilled in the art and are intended to bewithin the scope of the following claims.

1. A method for the collection and maintenance of detectability of aplurality of species of microbiological agents selected from the groupconsisting of bacteria, fungi, and viruses, in a primary gynecologicalsample and for handling a plurality of primary gynecological samples andmanaging information associated therewith for reporting a sum ofdiagnostic results for each primary gynecological sample, comprising thesteps of: (a) collecting a primary gynecological sample from a patient,(b) providing a transport medium in a resealable container withinstructions for preparation and handling of said primary gynecologicalsample and an indication of the detectability of the plurality ofspecies contained in said primary gynecological sample, wherein saidtransport medium is BD Cellmatics Viral Transport Medium®; (c) addingsaid primary gynecological sample to said transport medium, wherein saidtransport medium supports viability of said plurality of species ofmicrobiological agents sustained at room temperature for at least 48hours and up to 5 days; (d) receiving a plurality of said primarygynecological samples in step (c), each primary gynecological samplehaving identity and a test requisition form containing informationassociated therewith, wherein said information indicates a test for atleast one species from a plurality of species; (e) entering saidinformation into a system to create a requisition file for each primarygynecological sample; (f) processing said information to create a listof each primary gynecological sample to be tested for each species; (g)dispensing an aliquot corresponding to each primary gynecological sampleinto an individual vessel, to create a secondary gynecological sample,for each designated test for different species on each secondarygynecological sample; (h) assembling a general supply of master reagentmix for each different test; (i) combining an aliquot of master reagentmix for each test with each corresponding secondary gynecological sampleto produce a diagnostic test reaction for each secondary gynecologicalsample; (j) determining the presence or absence of a certain product ofeach reaction to produce a result, recording said result of eachreaction; (k) combining said result of each reaction derived from eachprimary gynecological sample into the requisition file for each primarygynecological sample on the system, thereby producing a sum of resultsfor each primary gynecological sample; and (l) reporting said sum ofresults to an attending physician who ordered the test within 24-48hours after receipt of said primary gynecological sample in step (d),wherein the plurality of species comprises at least one species selectedfrom the group consisting of Molluscum contagiosum virus, Mycoplasmagenitalium, Mycoplasma hominis, Candida dubliniensis, Candida krusei,Candida lusitaneae, Atopobium vaginae, erythromycin-resistantStreptococcus agalactiae, clindamycin-resistant Streptococcusagalactiae, Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31, HPV-33,HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66,HPV-68, HPV-6/11, HPV-42, HPV-43, and HPV-44.
 2. The method according toclaim 1 wherein at least one species within the plurality of species isindicative of at least one gynecological disorder.
 3. The methodaccording to claim 2 wherein at least one (1) species of the pluralityof species is selected from the group consisting of Gardnerellavaginalis, Molluscum contagiosum Virus, Mycoplasma genitalium, andMycoplasma hominis.
 4. A method for the collection and maintenance ofdetectability of a plurality of species of microbiological agentsselected from the group consisting of bacteria, fungi, and viruses, in aprimary gynecological sample and for handling a plurality of primarygynecological samples and managing information associated therewith forreporting a sum of diagnostic results for each primary gynecologicalsample, comprising the steps of: (a) providing a kit comprised of atransport medium in a resealable container, a sterile swab, andinstructions for preparation and handling of a primary gynecologicalsample and a writing which indicates the detectability of the pluralityof species, wherein said transport medium is BD Cellmatics ViralTransport Medium®, and wherein viability of said plurality of species ofmicrobiological agents is sustained at room temperature for at least 48hours and up to 5 days; (b) adding a collected primary gynecologicalsample to said transport medium in said kit, wherein said transportmedium supports viability of said plurality of species ofmicrobiological agents sustained at room temperature for at least 48hours and up to 5 days; (c) receiving a plurality of said primarygynecological sample in said transport medium, each such sample beingderived from a kit as recited in (a), having identity and a testrequisition form containing information associated therewith, whereinsaid test requisition information indicates a test for at least onespecies from the plurality of species; (d) entering said informationinto a system to create a requisition file for each primarygynecological sample; (e) processing said information to create a listof primary gynecological samples to be tested for each species; (f)dispensing an aliquot corresponding to each primary gynecological sampleinto an individual vessel, to create a secondary gynecological sample,for each designated test for different species on each gynecologicalsample; (g) assembling a general supply of master reagent mix for eachdifferent test; (h) combining an aliquot of master reagent mix for eachtest with each corresponding secondary gynecological sample to produce adiagnostic test reaction for each secondary gynecological sample; (i)determining the presence or absence of a certain product of eachreaction to produce a result, recording the result of each reaction; (j)combining the result of each reaction derived from each primarygynecological sample into the requisition file for each gynecologicalsample on the system, thereby producing a sum of results for eachprimary gynecological sample; and (k) reporting the results to anattending physician who ordered the test within 24-48 hours afterreceipt of said primary gynecological sample in step (c), wherein theplurality of species comprises at least one species selected from thegroup consisting of Molluscum contagiosum virus, Mycoplasma genitalium,Mycoplasma hominis, Candida dubliniensis, Candida krusei, Candidalusitaneae, Atopobium vaginae, erythromycin-resistant Streptococcusagalactiae, clindamycin-resistant Streptococcus agalactiae,Lymphogranuloma venereum, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35,HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68,HPV-6/11, HPV-42, HPV-43, and HPV-44.
 5. The method according to claim 4wherein the presence or absence of a product of at least one reaction iselectronically recorded by means of the system.
 6. The method accordingto claim 4 wherein at least one diagnostic test reaction comprises areal-time Polymerase Chain Reaction (PCR).
 7. The method according toclaim 4 wherein the results are reported within about fifty (50) twentyfour (24) hours of receiving the sample.
 8. The method according toclaim 4 wherein the results are reported within about thirty (30) hoursof receiving the sample.
 9. The method according to claim 4 whichcomprises pyrosequencing.
 10. A method for the collection andmaintenance of detectability of a plurality of species ofmicrobiological agents of bacteria, in a primary gynecological sampleand for handling a plurality of primary gynecological samples andmanaging information associated therewith for reporting a sum ofdiagnostic results for each primary gynecological sample, comprising thesteps of: (a) providing a transport medium in a resealable containerwith instructions for preparation and handling of a primarygynecological sample and an indication of the detectability of theplurality of species, wherein said transport medium is BD CellmaticsViral Transport Medium®, and wherein viability of said plurality ofspecies of microbiological agents is sustained at room temperature forat least 48 hours and up to 5 days; (b) receiving a plurality of saidprimary gynecological samples, each primary gynecological sample havingidentity and a test requisition form containing information associatedtherewith, wherein the test requisition information indicates a test forat least one species from a plurality of species; (c) entering theinformation into a system to create a requisition file for each primarygynecological sample; (d) processing the information to create a list ofeach primary gynecological samples to be tested for each species; (e)dispensing an aliquot corresponding to each primary gynecological sampleinto an individual vessel, to create a secondary gynecological sample,for each designated test for different species on each secondarygynecological sample; (f) assembling a general supply of master reagentmix for each different test; (g) combining an aliquot of master reagentmix for each test with each corresponding secondary gynecological sampleto produce a diagnostic test reaction for each secondary gynecologicalsample; (h) determining the presence or absence of a certain product ofeach reaction to produce a result, recording the result of eachreaction; (i) combining the result of each reaction derived from eachprimary gynecological sample into the requisition file for each primarygynecological sample on the system, thereby producing a sum of resultsfor each primary gynecological sample; and reporting the results to anattending physician who ordered the test within 24-48 hours afterreceipt of said primary gynecological sample in step (b) wherein theplurality of species comprises at least one species selected from thegroup consisting of Atopobium vaginae, erythromycin-resistantStreptococcus agalactiae, clindamycin-resistant Streptococcusagalactiae.